G-protein-coupled receptors (GPCRs) are the most widely targeted proteins for therapeutic purposes. Structurally, this class of proteins comprises an extracellular N terminal region and an intracellular C terminal region, which are joined by a transmembrane (TM) region comprising seven alpha-helical domains that traverse the cellular membrane bilayer.
The function of each domain (the N terminus and the C terminus) of the GPCRs has been elucidated. Each of the domains of a GPCR has a distinct function. More particularly, GPCRs retain all of their known ligand binding regions within the extracellular regions and TM domains 2 through 7 (e.g., Ling et al., (1999) Proc. Natl. Acad. Sci. U.S.A. 96:7922-7927; Gardella & Juppner, (2001) Trends Endocrin. Metab. 12(5):210-217; Vaidehi et al., (2002) Proc. Natl. Acad. Sci. U.S.A. 99:12622-12627), while the intracellular regions regulate the cell signalling and receptor internalization functions (Trejo et al., (1998) Proc. Natl. Acad. Sci. U.S.A. 95:13698-13702; Heding et al., (1998) J. Biol. Chem. 273(19):11472-11477; Trejo &. Coughlin, (1999) J. Biol. Chem. 274(4):2216-2224; Castro-Fernandez & Conn, (2002) Mol. Cell. Endocrinol. 191:149-156). G-protein coupled receptors, including chemokine receptors, have a wide range of specificities in terms of the signals they receive and those they transduce. As described further herein, these observations have been applied to the present invention in the form of chemokine receptor chimeras that retain ligand binding ability as well as G-protein-mediated signalling activity.
The chemokine receptor family of G-protein coupled receptors represents the largest group of peptide-binding GPCRs described to date (Onuffer & Horuk, (2002) Trends Pharmacol. Sci. 23(10):459-467). In this capacity, the bound peptides are chemokines for the chemokine receptor (the GPCR) (see, e.g., Yoshie et al., (2001) Adv. Immunol. 78:57). Chemokines are about 4 to about 14 kDa in size and comprise four conserved cysteine residues. They are broadly grouped into two groups: a major group comprising the CC and CXC subgroups in which two cysteines are adjacent or separated by one residue, and a minor group comprising the C and CXXXC subgroups in which the second cysteine is absent or is separated from the first cysteine by three residues (see, e.g., Horuk, (2003) Methods 29:369-375; Horuk, (2001) Cytokine Growth Factor Rev. 12:313-335). The classification scheme depends on the number and position of the first two conserved cysteine residues (Horuk, (2003) Methods 29:369-375).
At least 18 chemokine receptors have been identified, including 10 CC-type chemokine receptors (CCR1 (Neote et al., (1993) Cell 72:415-425; Gao et al., (1993) J. Exp. Med. 177:1421-1427), CCR2 (Charo et al., (1994) Proc. Natl. Acad. Sci. U.S.A. 91:2752-56), CCR3 (Combadiere et al., (1995) J. Biol. Chem. 270:16491-16494; Combadiere et al., (1995) J. Biol. Chem. 270:30235; Daugherty et al., (1996) J. Exp. Med. 183:2349-2354; Ponath et al., (1996) J. Clin. Invest. 97:604-612), CCR4 (Power et al., (1995) J. Biol. Chem. 270:19495-19500), CCR5 (Samson et al., (1996) Biochem. 35:3362-3367; Combadiere et al., (1996) J. Leukocyte Biol. 60:147-152), CCR6 (Baba et al., (1997) J. Biol. Chem. 272:14893-14898), CCR7 (Yoshida et al., (1997) J. Biol. Chem. 272:13803-13809), CCR8 (Tiffany et al., (1997) J. Exp. Med. 186:165-170; Roos et al., (1997) J. Biol. Chem. 272:17251-17254; Horuk et al., (1998) J. Biol. Chem. 273:386-391; Goya et al., (1998) J. Immunol. 160:1975-1981), CCR9 (Zaballos et al., (1999) J. Immunol. 162:5671-5675), CCR10 (Homey et al., (2000) J. Immunol. 164:3465-3470; Jarmin et al., (2000) J. Immunol. 164:3460-3464)) and 8 of the CXC, CXXXC and XC types (CXCR1 (Holmes et al., (1991) Science 253:1278-1280), CXCR2 (Murphy & Tiffany, (1991) Science 253:1280-1283), CXCR3 (Marchese et al., (1995) Genomics 29:335-344; Loetscher et al., (1996) J. Exp. Med. 184:963-969), CXCR4 (Nomura et al., (1993) Int. Immunol. 5:1239-1249; Federspiel et al., (1993) Genomics 16:707-712; Jazin et al., (1993) Regul. Pep. 47:247-258; Herzog et al., (1993) DNA Cell Biol. 12:465-471; Loetscher et al., (1993) J. Biol. Chem. 269:232-237), CXCR5 (Legler et al., (1998) J. Exp. Med. 187:655-660), CXCR6 (Matloubian et al., (2000) Nature Immunol. 1:298-304), CXXXCR1 (Combadiere et al., (1998) J. Biol. Chem. 273:23799-23804) and XCR1 (Yoshida et al., (1998) J. Biol. Chem. 273:16551-16554)). See, e.g., Horuk, (2001) Cytokine Growth Factor Rev. 12:313-335 for a review of chemokine receptors.
The nucleic acid sequences encoding the known chemokine receptors (which can be employed in the present invention and are incorporated herein by reference) are publicly available from the GenBank database and have the following accession numbers:
Table of GenBank AccessionsCCR1L10918, L09230, D10925CCR2U03905, U80924, D29984, U03882, U80924CCR3U28694, U51241, U49727CCR4X85740CCR5U54994, X91492, U57840CCR6U60000, U45984, U68032, Z79782CCR7X84702, L31581, L31584, L08176CCR8U45983, Z79782, U62556, Y08456CCR9U45982CCR10U13667CXCR1L19591, U11870, X65858, L19592, M68932CXCR2L19593, M94582, U11869, M73969, M99412CXCR3X95876, U32674CXCR4X71635, D10924, M99293, L01639, L06797CXCR5X68149, X68829CXCR6U73531CX3CR1U20350, U28934XCR1L36149
A function of the chemokine receptor/chemokine combination is to attract and activate cells involved in a variety of immune responses (see, e.g., Yoshie et al., (2001) Adv. Immunol. 78:57; Rollins, (1997) Blood 90:909; Baggiolini, (1998) Nature 392:565; Nagasawa et al., (1996) Nature 382:635). Depending on the cellular distribution and patterns of expression/production of these pairs of proteins, the coordination of extremely complex biological, notably immunological, phenomena can be accomplished (Horuk, (2001) Cytokine Growth Factor Rev. 12:313-335; Baggiolini, (1998) Nature 392:565-68). Because multiple chemokine receptors are often expressed in a single cell type, and because many chemokine receptors are capable of binding multiple chemokines, the complexity of possible interactions is enormous.
The only known receptor for the chemokine eotaxin is CCR3; however, CCR3, (the eotaxin receptor) is also capable of binding other chemokines including eotaxin-2, RANTES, MCP-2, MCP-3 and MCP-4 (see, e.g., Horuk, (2001) Cytokine Growth Factor Rev. 12:313-335; Baggiolini, (1998) Nature 392:565-68). In the case of CCR3 and its peptide ligand, eotaxin, the distribution and expression patterns of these two proteins suggest a role in an inflammatory process related to asthma (Baggiolini et al., (1997) Annu. Rev. Immunol. 15:675-705) and to contact dermatitis (Taha et al., (2000) J. Allergy Clin. Immunol. 105:1002-1007; Yawalkar et al., (1999) J. Invest. Dermatol. 113:43-48; Ying et al., (1999) J. Immunol. 163:3976-3984). In one example of such a process, following functional binding, CCR3 ligands stimulate calcium flux, actin reorganization, integrin upregulation, receptor internalization, activation of signal transduction pathways and cell migration (Adachi et al., (2001) J. Immunol. 167:4609-4615; El-Shazly et al., (1999) Biochem. Biophys. Res. Comm. 264(1):163-170; Elsner et al., (1996) Eur. J. Immunol. 26:1919-1925; Elsner et al., (1998) Eur. J. Immunol. 28:2152-2158; Kampen et al., (2000) Blood 95:1911-1917; Lundahl et al., (1998) Inflammation 22:123-135; Tachimoto et al., (2002) Am. J. Respir. Cell Mol. Biol. 26: 645-649; Tenscher et al., (1996) Blood 88: 3195-3199; Woo et al., (2000) Biochem. Biophys. Res. Comm. 298: 392-397; Zimmerman et al., (1999) J. Biol. Chem. 274: 12611-12618). The eotaxin receptor is known to be restricted in expression primarily to eosinophils, T-helper cells of the TH2 variety (Sallusto et al., (1997) Science 277:2005-2007), basophils (Uguccioni et al., (1997) J. Clin. Invest. 100:1137-43), mast cells, platelets, dendritic cells and based upon worked described herein, also monocytes. For the most part these cell types are known to be associated with a variety of acute and chronic allergic reactions including resistance to certain parasitic infections.
Continuing, it is known that anti-CCR3 antibody administered to mice by both intraperitoneal, and intra-nasal routes abrogates the eosinophil recruitment into the lung following intra-nasal allergen challenge (Justice et al., (2003) Am. J. Physiol. Lung Cell Mol. Physiol. 284:L169-L178). The antibody also eliminates airway hyper-responsiveness to methacholine challenge. CCR3 knockout mice exhibit markedly reduced eosinophil recruitment to the lung and skin following allergen challenge (Humbles et al., (2002) Proc. Natl. Acad. Sci. U.S.A. 99(3): 1479-84; Ma et al., (2002) J. Clin. Invest. 109(5):621-28). The effect on airway hyper-responsiveness, however, depends on the route of antigen sensitization. Mice sensitized by intraperitoneal injection show a slightly enhanced hyper-responsiveness (Humbles et al., (2002) Proc. Natl. Acad. Sci. U.S.A. 99(3): 1479-84), whereas those sensitized by epi-cutaneous application of antigen show a near-complete reduction of hyper-responsiveness.
CCR2 exists in two isoforms, CCR2A and CCR2B (Charo et al., (1994) Proc. Natl. Acad. Sci. U.S.A. 91:2752-56). In contrast to CCR3, which is primarily expressed in eosinophils, CCR2 (also referred to as MCP-1R) is predominantly expressed in the monocyte/macrophage lineage and its primary peptide ligand, MCP-1, is thought to act to recruit monocytes to sites of inflammation (Taub et al., (1993) Science 260:355; Roth et al., (1995) Eur. J. Immunol. 25:3482). In addition to MCP-1(α nd β), chemokine ligands known to interact with CCR2 include MCP-2, MCP-3, MCP-4 and MCP-5. Expression of CCR2 has also been described on basophils, NK cells, memory T cells, eosinophils and dendritic cells. Following functional binding, CCR2 ligands stimulate calcium flux, actin reorganization, integrin upregulation, receptor internalization, activation of signal transduction pathways and cell migration. Based upon the cellular distribution and activity of CCR2 and its ligands, therapeutic potential of CCR2 inhibitors may exist for chronic inflammatory conditions, such as atherosclerosis, rheumatoid arthritis and multiple sclerosis.
In the case of CCR2A (GenBank Accession No. AF545480), it is predicted that residues 1-42 of SEQ ID NO:2 comprise the N terminal region, that residues 310-374 of SEQ ID NO:2 comprise the C terminal region and that residues 43-309 of SEQ ID NO:2 comprise the TM region. In the case of CCR2B (GenBank Accession No. U03905), it is predicted that residues 1-42 of SEQ ID NO:4 comprise the N terminal region, that residues 310-360 of SEQ ID NO:4 comprise the C terminal region and that residues 43-309 of SEQ ID NO:4 comprise the TM region. With respect to CCR3 (GenBank Accession No. U28694), it is predicted that residues 1-34 of SEQ ID NO:6 comprise the N terminal region, that residues 306-355 of SEQ ID NO:6 comprise the C terminal region and that residues 35-305 of SEQ ID NO:6 comprise the TM region. The TM region creates extensive intracellular and extracellular loops of protein that confer specific biological reactivity and function. GPCRs are capable of responding to a wide variety of stimuli including light, odorants, ions, lipids, peptides and globular proteins.
The present invention relates to chimeric chemokine receptors. Several particular chimeric chemokine receptors have previously been generated. For example, Peiper et al. report the generation of (1) a chimera comprising the N terminal region of DARC, which was joined with the remaining portion of CCR1; and (2) a chimera comprising the N terminal region of CCR1, which was joined with the remaining portion of DARC (Peiper et al., (1997) Method Enzymol. 288:57-71). The chimeras of Peiper et al., however, only incorporated the extracellular N terminal region of CCR1 and DARC. Further, Alkhatib et al. (Alkhatib et al., (1997) J. Biol. Chem. 33:20420-26) and Pease et al. (Pease et al., (1998) J. Biol. Chem. 273(32):19972-76) prepared various CCR1/CCR3 chimeras. The chimeras of Alkhatib and Pease, however, also include only the extracellular N terminus of a CCR1 or CCR3 receptor joined with the remainder of a CCR3 or CCR1 receptor, respectively. Hill et al. prepared chimeras comprising the N terminal domain of CCR1, CCR2 and CXCR4 and the remaining portion of CCR5, but again, these chimeras did not incorporate a contiguous N terminus/seven TM helix element (Hill et al., (1998) Virol. 248:357-71). Similarly, Rucker et al. prepared CCR2B/CCR5 chimeras (Rucker et al., (1996) Cell 87:437-46), but these chimeras did not incorporate an intact N terminus/seven TM helix element from a single receptor. Chimeras comprising non-chemokine receptor components have also been prepared (CCR2/CD8; Monteclaro & Charo, (1997) J. Biol. Chem. 272(37):23186-90) as well as chimeras formed between orthologs of the same protein (human/macaque CCR3; Sol et al., (1998) Virol. 240:213-20), but neither of these chimeras incorporated an intact N terminus/seven TM helix element from a single receptor.
Although at least the above described chimeric receptors have been generated, these chimeras do not comprise a contiguous N terminus/seven TM helix element in general, nor do they comprise a region of a CCR3 receptor joined with a region of a CCR2 receptor in particular. Therefore these chimeras do not fully exhibit the properties of both of the CCRs that were used to construct the chimera (i.e., the properties of the N terminal and TM region of a first CCR, such as CCR3 and the intracellular C terminus of a second CCR, such as CCR2). Notably, these previously-generated chimeras do not include the TM region of the N terminal component of the chimera.
An impetus for generating the chimeric receptors of the present invention was the need for a receptor capable of high-affinity binding to the cognate ligand while retaining at least some of the downstream signalling capabilities associated with the native receptor. Such a chimera could be employed in a screening assay to identify chemokines that bind to a chemokine receptor and/or induce signalling. Prior to the present invention, such a chimeric receptor was lacking in the art.
Thus, what is needed is a chimeric chemokine receptor comprising the N-terminus through at least the last residue of the seventh transmembrane region of a first CCR (e.g., CCR3 or CCR2) joined to all or a portion of the C-terminus of a second CCR (e.g., CCR2 or CCR3, respectively). Such a receptor would facilitate a number of different assays, such as more accurate chemokine ligand binding, and signalling assays than can be achieved by employing the chimeras known in the art. Further, by modifying just the cytoplasmic tail of a chemokine receptor it may also be possible to generate alternative signalling pathways upon binding of the cognate ligand. These alternative pathways might prove to be easier to describe and/or quantify than those pathways associated with the wild-type receptor. The chimeric chemokine receptor would also be useful in modulator design efforts. The present invention solves these and other problems.